PDBsum entry 3c2m

Transferase, lyase/DNA

PDB id: 3c2m

Contents

Protein chain

326 a.a.

DNA/RNA

Ligands

EDO ×2

F2A

Metals

_NA ×6

_MN ×7

Waters ×307

References listed in PDB file

Key reference

Title

Structures of DNA polymerase beta with active-Site mismatches suggest a transient abasic site intermediate during misincorporation.

Authors

V.K.Batra, W.A.Beard, D.D.Shock, L.C.Pedersen, S.H.Wilson.

Ref.

Mol Cell, 2008, 30, 315-324. [DOI no: 10.1016/j.molcel.2008.02.025]

PubMed id

18471977

Abstract

We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.

Figure 1.
Figure 1. Closed Conformation of the Ternary Substrate Complex with an Active-Site Mismatched Nascent Base Pair
(A) Licorice representation of the Pol β backbone of the binary DNA complex (PDB ID 1BPX; orange) and ternary substrate complex (green) with an incorrect incoming nucleotide (dAMPCPP; yellow carbons) with a templating guanine. The catalytic and DNA-binding subdomains superimposed (gray backbone) with an rmsd of 0.56 Å (177 Cα). The DNA is omitted for clarity, but the 5′→3′ direction of the primer entry into the active site is indicated with a solid arrow. The open and closed positions of α helix N are shown. The amino-terminal lyase domain and carboxyl-terminal N subdomain (colored) move in response to binding an incorrect dNTP. The amino- (N) and carboxyl-terminal (C) ends are labeled.
(B) Ribbon representation of the Pol β backbone of the ternary substrate complex with correct (gray) or incorrect (green) incoming nucleotides. The polymerase domains with a correct (dA-dUMPNPP; PDB ID 2FMS) and incorrect (dG-dAMPCPP) nascent base pair were superimposed with an rmsd of 0.62 Å (314 Cα). The superimposed structures indicate that α helix N is in a “closed” position like that observed with a Watson-Crick nascent base pair. The incoming dAMPCPP of the mismatch structure is shown (yellow carbons), but the DNA is omitted for clarity. The amino terminus (N) of the lyase domain is also indicated.

Figure 4.
Figure 4. Position of Key Protein Residues in Closed Polymerase Complexes with Correct or Incorrect Incoming Nucleotides
(A) Two views (major groove view, top; −90° rotation of the top view, bottom) of the nascent base pair of the mismatch structure (dG-dAMPCPP; green carbons) superimposed (see Figure 1) with that for a correctly matched base pair (gray carbons). The templating base is omitted from the correctly matched overlay for clarity. The structure illustrates the position of key protein side chains that can influence catalytic behavior. For catalytic activation with the correct incoming nucleotide, N subdomain closing is associated with the loss of a salt bridge between Arg258 (R258) and Asp192 (D192), which coordinate both active-site metals (M^2+), and the formation of hydrogen bonds (black dashed lines) with Glu295 (E295) and Tyr296 (Y296). Phe272 (F272) is repositioned in the closed complex to insulate Asp192 from Arg258. Arg283 (R283) that is situated in the N subdomain interacts with the minor groove edge of the templating strand (not shown). With an incorrect incoming nucleotide, R258 and R283 are in conformations that preclude catalytic activation. Arg283 is observed to hydrogen bond with the minor groove edge and phosphate backbone of the templating base. Other key residues (Asp192, Asn279, and Phe272) are observed in similar positions as that found with a correct incoming nucleotide.
(B) Major groove view of the nascent base pair of the dG/dC-dAMPCPP superimposed mismatch structures (dC template, light blue carbons; dG template, green carbons).

The above figures are reprinted from an Open Access publication published by Cell Press: Mol Cell (2008, 30, 315-324) copyright 2008.